Project Summary Despite significant advances in breast cancer detection and therapy, up to 30% of women diagnosed with early-stage disease are at risk of recurrence and death. Adjuvant endocrine and chemotherapy, and HER2- targeted therapy with trastuzumab monoclonal antibody, which is a standard treatment for patients with HER2- positive disease (~20% of all cases), significantly improve the outcome. Unfortunately, approximately half of HER2-positive patients do not benefit from trastuzumab or have disease that becomes refractory to it. Chemotherapy alone is of little benefit in this setting, and novel treatments that can overcome trastuzumab resistance are being developed. The trastuzumab-mertansine antibody-drug conjugate (ADC), T-DM1 (Genentech), demonstrates high efficacy in trastuzumab-refractory disease. However, the selection of patients for T-DM1 therapy is currently based on the assessment of HER2 lesion(s) status by biopsy, which may not provide reliable results for heterogeneous disease. In addition, the long circulation half-time of cytotoxic ADC can result in systemic toxicity. In this application, we propose to develop and validate an image-guided, two- component therapeutic system for targeted treatment of HER2-positive breast cancer that takes advantage of bioorthogonal in vivo click chemistry and can address the concerns related to ADC-based therapy. The central hypothesis is that an image-guided delivery system, based on the pretargeting of HER2 receptors with a bispecific antibody and cytotoxic nanocarriers, will provide real-time, noninvasive assessment and phenotyping of the disease, as well as high efficacy and reduced systemic toxicity for the treatment of trastuzumab-resistant HER2+ breast cancers. The main advantages are: (i) efficient labeling of cancer cells with a non-toxic pretargeting antibody component; (ii) high affinity binding and rapid internalization of the therapeutic cytotoxic component by pre-labeled cancer cells; (iii) a short circulation time and rapid clearance of the therapeutic component ensures low systemic toxicity; and (iv) image guidance enables selection of tumors/patients for therapy, verification of pre-labeling efficiency, and optimization of the administration time for the therapeutic component. Our preliminary studies with HER2+ breast cancer models demonstrated high efficacy and low toxicity of the pretargeting therapy, with complete remission achieved in several animals. The radiolabeled delivery components will be synthesized, characterized, and tested in preclinical models of HER2+ breast cancer in immunodefficient and immunocompetent animals. The treatment efficacy and toxicity of the pretargeting therapy will be compared to the gold standard, T-DM1. We envision that the pretargeting image-guided therapy will provide high efficacy combined with minimal toxicity. Humanized antibody, human protein carriers, bioorthogonal click chemistry, and clinical modalities used for image-guidance will ensure a translational path for the system with a future potential use in patients with recurrent metastatic breast cancer, using image-guidance to noninvasively assess HER2 expression and targeting in heterogeneous disease.